GSM7713221: LIGHTCART_T0_ADT_HTO; Homo sapiens; OTHER
Cells were stained with Trypan blue and Countess II Automated Cell Counter (ThermoFisher) was used to assess both cell number and viability. Following QC, the single cell suspension was loaded onto Chromium Next GEM Chip K (10X Genomics PN 1000286) and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 10,000 cells proceeded using the Chromium Next GEM Single Cell 5' Kit v2 (10X Genomics PN 1000263) according to the manufacturer's protocol. cDNA amplification included 13 cycles and 50 ng of the material was used to prepare sequencing libraries with 14 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/88 run using the NovaSeq 6000 S4 Reagent Kit (200 cycles) (Illumina). An average of 22,500 reads was generated per cell. Amplification products generated using the methods described above included both cDNA and feature barcodes tagged with cell barcodes and unique molecular identifiers. Smaller feature barcode fragments were separated from longer amplified cDNA using a 0.6X cleanup using aMPure XP beads (Beckman Coulter catalog # A63882). Libraries were constructed using the 5' Feature Barcode Kit (10X Genomics PN 1000256) according to the manufacturer's protocol with 8 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/88 run using the NovaSeq 6000 S4 Reagent Kit (200 cycles) (Illumina). An average of 82 million paired reads was generated per sample.